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Figure 8


Fig. 8. Trafficking of VSVG-GFP from the Golgi to the plasma membrane is inhibited upon interfering with interactions between dynamin II and syndapin II. (A,B) COS-7 cells transfected with a temperature-sensitive VSVG-GFP showed a strong perinuclear fluorescence after 15 minutes at 32°C (A; start of chase), which declined as fluorescent material was transported to the plasma membrane after 90 minutes at 32°C (B). Magnifications of boxed areas in A and B display the region of interest (gray circle) used for the quantitative image analyses (I). (C,D) Cells co-overexpressing Myc-tagged dynamin II{Delta}PRD (Dyn II{Delta}PRD) displayed perinuclear accumulations of VSVG-GFP at the start of chase (C) and after 90 minutes at 32°C (D). (E,F) Cells overexpressing the Xpress-tagged syndapin II SH3 domain also show an arrest of VSVG-GFP in the perinuclear region (F). (G,H) By contrast, co-overexpression of full-length syndapin II-l had no negative effect on Golgi-to-PM transport but VSVG-GFP was observed at the plasma membrane after 90 minutes (H). Images were processed by Adobe Photoshop to visualize clearly the different VSVG-GFP distributions. Co-transfected cells are marked by asterisks. Bar, 20 µm. (I) Averaged fluorescence intensities within the perinuclear region of interest after 90 minutes at 32°C expressed as percentage of start values. Fluorescence was measured as 8-bit gray values by unbiased experimenters. 255 corresponds to white, 0 to black. Background values were subtracted. Control (- -): start of chase, 275 cells; 90 minutes, 280 cells. Dynamin (Dyn) II{Delta}PRD: start, 83 cells; 90 minutes, 82 cells. Syndapin II SH3 domain (Sdp II SH3): start, 57 cells; 90 minutes, 32 cells. Syndapin II-l full-length (Sdp II-I): start, 66 cells; 90 minutes, 52 cells. Error bars represent standard deviations between independent data sets.





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